Geranium oil and  constituents thereof for treatment of neurodegenerative diseases

ABSTRACT

The invention relates to the use of effective amount of  Pelargonium graveolens  essential oil or extract or constituents thereof selected from a group consisting of: (S)(−)citronellol, linalool, menthone and isomenthone or any combination thereof, in the preparation of a medicament for treating a mammal suffering from or susceptible to a neurodegenerative condition which can be improved or prevented by inhibition of acetylcholinesterase (ACliE).

FIELD OF THE INVENTION

The invention relates to geranium oil formulations for treatingneurodegenerative diseases, particularly to neurodegenerative diseaseswhich can be improved or prevented by inhibition of acetylcholinesterase(AChE).

DESCRIPTION OF THE STATE OF THE ART

Essential oils are a folk medicine and recently their use has expandedworldwide to include therapy against various kinds of inflammatorydiseases, such as allergy, rheumatism, arthritis and bronchitis. Theseactivities have mainly been recognized through clinical experience, buthave been little elucidated experimentally.

Geranium oil is one of the most important natural raw materials in thefragrance industry. It is used for creating rosy notes, especially insoap. The main cultivation areas are Reunion and Madagascar (Bourbontype), Morocco and Egypt (North African type), and China.

Geranium oil is obtained by steam distillation of the flowering herbPelargonium graveolens 1′Heritier ex Aiton, P. roseum Wilidenow, andother non-defined hybrids that have developed into different ecotypes indifferent geographical regions. The oil is amber to greenish-yellowliquid with the characteristic roselike odor of the plant.

Maruyama et al., Mediators Inflamm. 2006; 2006(3):62537 teach thatinjection of geranium oil suppressed the carrageenan-induced footpawedema and increase in tissue myeloperoxidase activity, and repeatedadministration of the oil suppressed collagen-induced arthritis.

According to Abe et al., Mediators of Inflammation, 13(1), 21-24(February 2004), leukocyte recruitment into the peritoneal cavity inmice was suppressed by intraperitoneal injections of Pelargoniumasperum.

US Publication 20080268078 reports the inhibition and/or reduction ofnitric oxide (NO) and/or prostaglandin E₂ synthesis, by administering ageranium plant extract comprises butylidene, phthalide, citronellol andgeraniol.

According to Elmann et al., Journal of Functional Foods 2 (2010) 17-22,the essential oil of geranium was tested for its anti-neuroinflammatoryeffects using primary cultures of microglial cells. Geranium oilinhibited NO production, as well as the expression of theproinflammatory enzymes cyclooxygenase-2 (COX-2) and induced nitricoxide synthase (iNOS) in primary cultures of activated microglial cells.

Parkinson's disease (PD) is the second most frequent degenerativedisorder after Alzheimer's disease. Clinically, its cardinal featuresinclude tremor, muscle rigidity, slowness of voluntary movement andpostural instability. Although PD neuropathology encompasses a number ofdifferent neurotransmitter pathways the disabling manifestations citedabove are attributed primarily to a deficit in brain dopamine (NatProtoc. 2007; 2(1):141-51). Among the different dopaminergic systems ofthe brain, the ascending nigrostriatal pathway has been consistentlyidentified as the most severely damaged in PD, underlined by the deathof the nigrostriatal dopaminergic neurons.

Alzheimer's disease is associated with a selective loss of cholinergicneurons in the brain (Davies and Maloney, Lancet. 1976 Dec. 25;2(8000):1403; Whitehouse et al., Ann Neurol. 1981 August; 10(2):122-6).The losses are accompanied by decreases in the neurotransmitteracetylcholine as well as in the respective enzymes that synthesize anddegrade acetylcholine, choline acetyltransferase andacetylcholinesterase (AChE) (Perry et al., Lancet. 1977 Jan. 22;1(8004):189; Bowen et al., J Neurol Sci. 1982 December;57(2-3):191-202). These observations stimulated a cholinergic hypothesisof Alzheimer's disease (Bartus et al., Science. 1982 Jul. 30;217(4558):408-14), which postulates that at least some of the cognitivedecline experienced by patients with the disease results from adeficiency in acetylcholine and thus in cholinergic neurotransmission.This hypothesis suggested that inhibitors of AChE might elevate levelsof acetylcholine in the brains of these patients and reverse thecognitive decline (Muramoto et al., Arch Neurol. 1979 August;36(8):501-3), and experimental evidence has supported this suggestion(Camps et al., Mol Pharmacol. 2000 February; 57(2):409-17).

Cholinesterase inhibitors, such as Donepezil®, Rivastigmine® andGalantamine®, are used as standard therapy for mild to moderateAlzheimer's disease and show therapeutic effect of this class ofmedications on the symptoms of Alzheimer's disease.

Umezu T, Phytother. Res. 26: 884-891 (2012) teaches that Pelargoniumgraveolens oil did not produce any effect on discrete shuttle-typeconditioned avoidance response in mice (abstract). The discreteshuttle-type conditioned avoidance response is useful for distinguishingCNS stimulants and CNS depressants.

It has now surprisingly been found that Pelargonium graveolens oil andconstituents thereof have good effect in preventing and improvingneurodegenerative symptoms.

SUMMARY OF THE INVENTION

The present invention relates to the use of P. graveolens essential oilsor extracts for the treatment of neurodegenerative diseases.

It has been found that essential oils of P. graveolens serve as AChEinhibitor. It has also been found that constituents of P. graveolensselected from the group consisting of (S)(−)-citronellol, linalool,menthone and isomenthone serve as AChE inhibitors.

The inventors have found that only the enantiomer (S)(−)citronellol butnot (R)(+)citronellol could inhibit the enzymatic activity of acetylcholinesterase. These results are quite unexpected when comparing themto the effect of the same enantiomers on Reactive Oxygen Species (ROS)in microglial cells. While both enantiomers show significant reductionon ROS production (FIG. 1), only the enantiomer (S)(−)citronellol showsinhibition of enzymatic activity of acetyl cholinesterase (FIGS. 2 and3).

The present invention relates to the use of effective amount of any oneof the Pelargonium graveolens constituents selected from a groupconsisting of: (S)(−)citronellol, linalool, menthone and isomenthone orany combination thereof, in the preparation of a medicament for treatinga mammal suffering from or susceptible to a neurodegenerative conditionwhich can be improved or prevented by inhibition of acetylcholinesterase(AChE).

The present invention further relates to the use of effective amount ofany one of the above Pelargonium graveolens constituents wherein saidneurodegenerative condition is Alzheimer's disease, Parkinson's disease,parkinsonian dementia (PDem), Parkinson's disease dementia (PDD),dementia with Lewy bodies (DLB), anorexia nervosa, traumatic braininjury (TBI), and Parkinson's disease falling short of dementia(CIND-PD).

The present invention further relates to Pelargonium graveolensessential oil for use in treating Parkinson's disease, parkinsoniandementia (PDem), Parkinson's disease dementia (PDD), dementia with Lewybodies (DLB), anorexia nervosa, traumatic brain injury (TBI), andParkinson's disease falling short of dementia (CIND-PD).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of enantiomers (S)(−)citronellol (FIG. 1A) and(R)(+)citronellol (FIG. 1B) of Geranium oil on the peroxylradical-induced oxidation of DCFH in primary microglial cells.

FIG. 2 shows the activity of acetyl cholinesterase after incubated withdifferent constituents of geranium oil and with geranium oil itself.

FIG. 3 shows the activity of acetyl cholinesterase after incubated withdifferent enantiomers of citronellol.

FIG. 4 shows the effect of Geraniol, Citronellol(+) and Citronellolformate on the activity of the human erythrocytes' acetylcholinesterase.

FIG. 5 shows the effect of geranium oil on body weights of MPTP-PD mice.

FIG. 6 shows the effect of geranium oil on Rota-Rod latency (FIG. 6A)and distance (FIG. 6B) of MPTP PD mice.

FIG. 7 shows the effect of geranium oil on social recognition ofMPTP-induced Parkinson's mice.

FIG. 8 shows the effect of geranium oil on the number of dopaminergicneurons in the substantia nigra.

DETAILED DESCRIPTION OF THE INVENTION

Essential oil or extract of P. graveolens or constituents thereofselected from the group consisting of (S)(−)-citronellol, linalool,menthone and isomenthone is expected to cause a significant increase inconcentrations of acetylcholine in the cerebral cortex and/orhippocampus and/or striatum and/or whole brain of a patient.Accordingly, therapeutic effect of various diseases associated with AChEinhibition and with an increase in concentrations of acetylcholine, maybe achieved by pharmaceutical compositions of the present invention.

Extracts of P. graveolens may be prepared using conventional methods.One exemplary extraction method is summarized below. In summary form,the method includes: (I) drying the selected plant parts and milling thedried plant parts to a fine powder; (2) mixing the plant powder to anextraction solvent; (3) removing the plant remnants by centrifugationand collecting the solvent supernatant; (4) removing the remainingsolvent and water in the supernatant by rotary evaporation and drying.One of ordinary skill in the art would realize that the method may bemodified appropriately, e.g., for the transition from a small-, orlaboratory-, scale to a commercial-scale method. The drying process maynot be needed for extraction purposes. Fresh plant material may bedirectly subjected to solvent extraction without the drying step. Driedplant parts do offer an advantage in terms of transport and storage ofthe crop; however, the activity of the extract from air-dried plants maybe lower than the activity of freeze-dried extracts due to possibleincrease in breakdown of components in the air-dried counterparts.

The extraction solvent may be a mildly polar fluid. The “mildly polar”fluid means a fluid that is slightly to moderately polar, as would beunderstood in the art. Mildly polar as used herein means moderatelyirregular distribution of electrons that is characterized by a weak toaverage degree of hydrophilicity. A mildly polar fluid includes allstraight chain and branched primary alcohols and chemical derivativesthereof, provided that the additional chemical groups do not destroy thepolarity of the fluid or increase the polarity of the fluid to the levelof water, which is expressly excluded from the definition of a mildlypolar fluid. Preferred mildly polar fluids are liquids, such as thelower molecular weight, straight chain, primary alcohols (e.g.,ethanol). Water is not a mildly polar fluid, but is a highly polaraqueous fluid. However, a mixture of water and a mildly polar fluid(e.g., ethanol) is itself a mildly polar fluid. An example of the latterfluid is 60% ethanol. A variety of mildly polar fluids, such asalcohols, may be used to extract efficacious materials from the selectedplant parts, including methanol, ethanol, and isopropanol. When alcoholis used, the resulting product is an ethanolic extract.

In a preferred embodiment, P. graveolens will be in the form ofessential oil. The essential oil may be either steam-distilled orhydro-distilled from the fresh herb foliage for one hour in a 130 Ldirect steam pilot plant apparatus or in a modified clevenger apparatusrespectively.

As already outlined, inhibitors of AChE might elevate levels ofacetylcholine in the brains of these patients and reverse theirbehavioral decline.

In a preferred embodiment, the plant essential oil or extract of P.graveolens is the sole active ingredient in a composition for treatingneurodegenerative disease. In another preferred embodiment, the plantessential oil or extract of P. graveolens is the sole active ingredientfor treating neurodegenerative disease.

In another embodiment, any combination of the active constituents thatis, only part of the active constituents (S)(−)citronellol, linalool,menthone and isomenthone are used for treating neurodegenerativedisease.

Choline-esterase inhibitors (such as donepezil, tacrine, rivastigmin andgalantamine) are widely used for improving cognition in Alzheimer'sdisease and show promising clinical result in Dementia with Lewy Bodies.

Pathology reports have shown that cholinergic forebrain neuronal lossesin parkinsonian dementia (PDem) are equal to or greater than that inAlzheimer disease (AD). Bohnen et al., Arch Neurol. 2003;60(12):1745-1748 shows that compared with controls, mean cortical AChEactivity was lowest in patients with PDem (−20.0%), followed by patientswith Parkinson disease without dementia (−12.9%; P<0.001). In vivocortical acetylcholinesterase (AChE) activity in healthy controlsubjects and in patients with mild AD, PDem, and Parkinson diseasewithout dementia using AChE positron emission tomography has beendetermined. Therefore, an embodiment of the invention is the use of P.graveolens essential oil or extract, (S)(−)-citronellol, linalool,menthone and isomenthone in the preparation of a medicament in thetreatment of parkinsonian dementia (PDem) and Parkinson disease withoutdementia.

Bohnen et al., J Neurol Neurosurg Psychiatry 2007; 78:641-643investigated the relationship between ratings of depressive symptoms andin vivo cortical acetylcholinesterase (AChE) activity in subjects withParkinson's disease (PD) and parkinsonian dementia (PDem). Subjects withPD and with PDem, underwent [¹¹C]methyl-4-piperidinyl propionate AChEpositron emission tomography imaging and clinical assessment includingthe Cornell Scale for Depression in Dementia (CSDD). Bohnen et al.concludes that depressive symptomatology is associated with corticalcholinergic denervation in PD.

According to Ballard et al., Drugs Aging. 2011 Oct. 1; 28(10):769-77,randomized controlled trials (RCTs) of the cholinesterase inhibitorrivastigmine have indicated modest but significant benefits incognition, function, global outcome and neuropsychiatric symptoms inboth Parkinson's disease dementia (PDD) and dementia with Lewy bodies(DLB). Therefore, another embodiment is the use of P. graveolensessential oil or extract, (S)(−)-citronellol, linalool, menthone andisomenthone in the preparation of a medicament in the treatment ofParkinson's disease dementia (PDD) and dementia with Lewy bodies (DLB).

Patients with anorexia nervosa present compromised affectivity,characterized by hypomanic, manic and depressive symptoms, and theircholinergic system is altered with a decrease in the release ofacetylcholine (Expert Opin. Investig. Drugs (2009) 18(5):569-571).Therefore, another embodiment of the invention is the use of P.graveolens essential oil or extract, (S)(−)-citronellol, linalool,menthone and isomenthone in the preparation of a medicament in thetreatment of anorexia nervosa.

Memory impairments following traumatic brain injury (TBI) are wellrecognized and can be difficult to treat, particularly for those TBIsurvivors who experience persistent amnesia. Morey et al., Brain Injury,VOL. 17, NO. 9, September 2003, 809-815 reports that theacetylcholinesterase inhibitor, Aricept® (donepezil hydrocholoride), maypotentially be useful in treating such memory problems. According toMorey et al., Aricept®, which increases the availability ofacetylcholine at the post-synaptic receptors by inhibitingacetylcholinesterase (AChE) function in the central nervous system hasbeen shown to be effective in treating symptoms of memory loss inpatients with AD and, more recently, with TBI. Therefore, anotherembodiment of the invention is the use of P. graveolens essential oil orextract, (S)(−)-citronellol, linalool, menthone and isomenthone in thepreparation of a medicament in the treatment of memory loss in patientswith AD and in the treatment of TBI.

According to Rolinski M et al. Cochrane Database Syst Rev. (2012),currently available evidence supports the use of cholinesteraseinhibitors in patient with Parkinson's disease dementia PDD andcognitive impairment in Parkinson's disease falling short of dementia(CIND-PD), with a positive impact on global assessment, cognitivefunction, behavioural disturbance and activities of daily living ratingscales. Therefore, another embodiment of the invention is the use of P.graveolens essential oil or extract, (S)(−)-citronellol, linalool,menthone and isomenthone in the preparation of a medicament in thetreatment of Parkinson's disease dementia (PDD) and CIND-PD.

Thus, according to the present invention, any neurodegenerativecondition susceptible of being improved or prevented by inhibition ofAChE may be under the scope of the invention.

FIG. 1 shows that constituents of Geranium oil (S)(−)citronellol and(R)(+)citronellol inhibit the peroxyl radical-induced oxidation of DCFHin primary microglial cells. Microglial cells were incubated for one hwith (S)(−)citronellol (FIG. 1A) and (R)(+)citronellol (FIG. 1B).Primary cultures of microglial cells has been prepared according toElmann et al., Journal of Functional Foods 2 (2010), pp. 18. As inElmann et al. BMC Complementary and Alternative Medicine 2011, 11:98,the cultures of microglial cells were then preloaded with thenon-fluorescent cell permeating compound, 2′7′-dichlorofluoresceindiacetate (DCF-DA) for 30 min and washed with PBS, after which,2,2′-Azobis(amidinopropane) (ABAP) (0.6 mM) was added and ROS levelswere measured at the indicated time points. Each point on the graphrepresents mean±SEM of 2 experiments (n=8).

As already mentioned, the inventors have unexpectedly found that onlythe enantiomer (S)(−)citronellol but not (R)(+)citronellol could inhibitthe enzymatic activity of acetyl cholinesterase. The unexpectedness ofthese results is demonstrated for example, when comparing the effect ofthe same enantiomers on Reactive Oxygen Species (ROS) in microglialcells. While both enantiomers show significant reduction on ROSproduction (FIG. 1), only the enantiomer (S)(−)citronellol showsinhibition of enzymatic activity of acetyl cholinesterase (FIGS. 2 and3).

The term “neurodegenerative condition or disease” is any condition ordisease of, relating to, or being a progressive loss of neurologicfunctions.

The term “treating” as used herein in relation to neurodegenerativediseases in a subject is intended to mean that the compound, essentialoil or extract or pharmaceutical composition of the invention reduces orabrogates the symptoms and/or cause of the neurodegenerative disease.

The term “prevention” as used herein in relation to neurodegenerativediseases in a subject is intended to mean that the compound, essentialoil or extract or pharmaceutical composition of the inventionsubstantially prevents and/or reduces the symptoms of theneurodegenerative diseases that would otherwise occur had the subjectnot been treated with the compound, essential oil or extract orpharmaceutical composition of the invention.

An “essential oil” is a product made by distillation with either wateror steam or by mechanical processing of citrus rinds or by drydistillation of natural materials. Following the distillation, theessential oil is physically separated from the water phase.

In all therapeutic applications and unless otherwise noted, “Pelargoniumgraveolens” means Pelargonium graveolens essential oil or extractwithout separating plant's active constituents. “Pelargonium graveolensconstituent” means a specific active constituent of Pelargoniumgraveolens.

Use of PET for Determining Whether Therapeutic Effects are Caused byInhibition of AChE.

The positron emission tomographic (PET) technology to measure AChEfunctional activity offers the prospect of determining cholinergicinnervation in vivo at the early stages of neurodegenerative disorders.For example acetyl cholinesterase (AChE) activity in the humanAD-affected brain has been mapped using PET and1-[¹¹C]methylpiperidin-4-yl propionate ([¹¹C]PMP) andN-[¹¹C]methylpiperidine-4-yl acetate radioligands (Bohnen et al., ArchNeurol. 2003; 60(12):1745-1748).

Therefore, for a neurodegenerative disease associated with decrease inlevels of acetylcholine, the PET technique, combined with known testsunique for that neurodegenerative disease for assessing the condition ofa subject may be used for determining whether improvement in a patient'scondition is a result of AChE Thus, this combined test (namely, (1) PETand (2) test for assessing the condition of a subject) may be madebefore and after administration of P. graveolens or constituentsthereof. For example, for assessing the relationship between ratings ofdepressive symptoms and in vivo cortical acetylcholinesterase (AChE)activity in subjects with parkinson's disease (PD) and parkinsoniandementia (PDem), the PET may be combined with the Cornell Scale forDepression in Dementia (CSDD). For TBI, the Token Test and the BostonNaming Test may be administered during baseline testing together withPET. For dementia with Lewy Bodies and Parkinson's disease Dementia, therandomized controlled trials (RCTs) of DLB and PDD may be used togetherwith PET. And, all of these combined tests may be made before and afteradministration of P. graveolens or constituents thereof.

The preparation of pharmaceutical compositions is known in the art, forexample as described in Remington's Pharmaceutical Sciences (18th ed.,Mack Publishing Co., Easton, Pa., 1990) and Pharmacopeia: NationalFormulary (Mack Publishing Company, Easton, Pa., 1984). For example, thecompound may be prepared into a variety of pharmaceutical compositionsin the form of, e.g., an aqueous solution, an oily preparation, a fattyemulsion, an emulsion, a gel, etc., and these preparations may beadministered as intramuscular or subcutaneous injection or as injectionto an organ, or as an embedded preparation or as a transmucosalpreparation through nasal cavity, rectum, uterus, vagina, lung, etc. Thecomposition may be administered in the form of oral preparations (forexample solid preparations such as tablets, capsules, granules orpowders; liquid preparations such as syrup, emulsions or suspensions).Compositions containing the compound may also contain a preservative,stabiliser, dispersing agent, pH controller or isotonic agent. Examplesof suitable preservatives are glycerin, propylene glycol, phenol orbenzyl alcohol. Examples of suitable stabilisers are dextran, gelatin,a-tocopherol acetate or alpha-thioglycerin. Examples of suitabledispersing agents include polyoxyethylene (20), sorbitan mono-oleate(Tween 80), sorbitan sesquioleate (Span 30), polyoxyethylene (160)polyoxypropylene (30) glycol (Pluronic F68) or polyoxyethylenehydrogenated castor oil 60. Examples of suitable pH controllers includehydrochloric acid, sodium hydroxide and the like. Examples of suitableisotonic agents are glucose, D-sorbitol or D-mannitol. The compositionmay also contain other constituents or additives such as apharmaceutically acceptable carrier, diluent, excipient, suspendingagent, lubricating agent, adjuvant, vehicle, delivery system,emulsifier, disintegrant, absorbent, preservative, surfactant, colorant,flavorant or sweetener, taking into account the physical and chemicalproperties of the compound being administered.

In another embodiment, P. graveolens essential oil is the sole activeingredient in a composition for treating neurodegenerative disease suchas Parkinson's disease.

In a yet another embodiment, any one of the Pelargonium graveolensconstituents selected from a group consisting of: (S)(−)citronellol,linalool, menthone and isomenthone or any combination thereof is/are thesole active ingredient in a composition for treating neurodegenerativedisease.

In another embodiment, the present invention provides pharmaceuticalcompositions for use in the treatment of Parkinson's disease. In anotherembodiment, the present invention provides pharmaceutical compositionsfor use in preventing deterioration of Parkinson's disease. Thisprevention is reflected by cessation of neuronal cells death in apatient showing Parkinson's disease symptoms. Results disclosed herewithfor prevention of neuronal cells death (see Example 7) is surprising.The direct and significant effect on the prevention of dopaminergicnerve cells was not expected by the inventors.

The MPTP Model

In order to demonstrate the therapeutic effect of P. graveolens intreating Parkinson's disease, the inventors have used the MPTP model.Among the various toxic models of PD, the MPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model has become the mostcommonly used. MPTP is the only known dopaminergic neurotoxin capable ofcausing a clinical picture in both humans and monkeys indistinguishablefrom PD. Second, although handling MPTP requires a series ofprecautions, its use is not otherwise technically challenging: it doesnot require any particular equipment such as a stereotaxic frame, nordoes it require surgery on live animals as for 6-hydroxydopamine orrotenone. And third, MPTP produces a reliable and reproducible lesion ofthe nigrostriatal dopaminergic pathway after its systemicadministration, which is often not the case for other documentedpoisons. MPTP causes parkinsonian features in humans and primates as aresult of dopaminergic degenerations in the nigrostriatal pathway. Itsneurotoxic effects also appear to involve energy depletion and freeradical generation. MPTP is converted to its metabolite MPP+ bymonoamine oxidase B (MAO-B). MPP+ is selectively accumulated by highaffinity dopamine transporters and taken up into the mitochondria ofdopaminergic neurons, where it disrupts oxidative phosphorylation byinhibiting complex I of the mitochondrial electron transport chain. Thisleads to impairment of ATP production, elevated intracellular calciumlevels, and free radical generation. Thereby exhibiting dopaminergicneurotoxicity. Therefore, MPTP is widely used as a tool to study themolecular events that lead to degeneration of dopaminergic neurons inanimal model of Parkinson's disease and to test potentialneuroprotective agents.

Preclinical Evaluation of Geranium (P. graveolens) Oil in the MPTPInduced Model of Parkinson's Disease in Mice.

In a demonstration of the present invention as applied to mice, thepreclinical efficacy of geranium (P. graveolens) oil was evaluated inthe 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced model ofParkinson's disease in C57BL/6 mice (The Parkinson's disease afflictedmice will be called from now on “MPTP PD mice”). MPTP was injected toC57BL/6 (11-13 mice/group) intraperitoneally (i.p.) daily during theinitial 5 days (day 1-5). Geranium oil was administered to MPTP PD miceon days 1-11 by oral gavage or by subcutaneouse injection. At the first5 days, the oil was administered 30 min before MPTP administration. Theeffect of geranium oil on motor coordination and cognitive function inexperimentally PD-induced mice was evaluated using the Rota Rod and thesocial recognition tests, respectively. At the end of the study, thesubjects' brains were removed and analyzed for neuronal cell loss in thesubstantia nigra.

As outlined, an accelerating rotarod is used to measure motorcoordination in mice. Mice have to keep their balance on a horizontalrotating rod, and rotation speed is accelerated from 4 to 20 rpm over 5min. A trial starts when the mouse is placed on rotating rod, and itstops when the mouse falls down or when 5 min are completed. Each mouseis experiencing 3 trials per day with an intertrial interval of 10 min.

EXAMPLES

All results disclosed in the Examples herewith referring to geraniumoil, have been obtained with the essential oil of the plant Pelargoniumgraveolens (P. graveolens).

Example 1 P. graveolens Constituents that Inhibit Acetyl CholinesteraseActivity

In this Example, Human erythrocytes acetyl cholinesterase (AChE) wasincubated with different concentrations of geranium oil or each of itsconstituents.

Preparation of Essential Oils and GC-MS Analyses.

P. graveolens was grown at the Newe Ya'ar Agricultural ExperimentalStation, under cultivated conditions. Seeds were taken from cooled seedstorage and were sown at Newe Ya'ar. The plant was cultivated in localclayey soil under drip irrigation, as described in Dudai et al., (2003),Flavour and Fragrance Journal, 18, 334-337. Plant samples of at least 5kg were steam-distilled in a 130 L direct steam pilot plant apparatusfor 1 h. The essential oils were diluted with petroleum ether and wereinjected to the a GC-MS, MSD (Agilent Technologies, Palo Alto, Calif.,USA) with an autosampler Combi Pal (CTC Analytics, Zwingen, Switzerland)equipped with a Rtx-5SIL MS (30 m×0.25 mm i.d.×0.25 lm) fused-silicacapillary column (Restek, Bellefonte, Pa., USA). Helium, at a constantpressure of 14.14 psi and a linear velocity of 47 cm⁻¹, was used ascarrier gas. The injector was kept at 250° C. and set for the 1:50 splitmode. The transfer line was kept at 280° C. The column was maintained at50° C. for 1 min, then programmed to 190° C. at 5° C. min⁻¹, then to260° C. at 10° C. min⁻¹. The MSD was operated in the electron ionizationmode at 70 eV, in the m/z range of 42-350. Identification of compoundswas performed by comparing their relative retention indices and massspectra with those of authentic samples, supplemented with the NIST 98and HPCH 2205 GC-MS libraries.

Enzymatic Activity.

In the results shown in FIG. 2, the different constituents wereincubated with human erythrocytes' acetyl cholinesterase (25 mU/mL). Theactivity of acetyl cholinesterase was determined using a commerciallyavailable kit (acetyl cholinesterase fluorescent activity assay kit,Arbor assays). FIG. 2 demonstrates that geranium oil,(S)(−)-citronellol, linalool, menthone and isomenthone inhibited theenzymatic activity.

Example 2 Difference Between Enantiomers of Citronellol on AChE Activity

Geranium oil contains the two enantiomers of citronellol [(R)(+):(S)(−)]in a ratio of ˜80:20. Thus, inventors have tested the effect of bothenantiomers on the enzymatic activity. As noted, only the enantiomer(S)(−)citronellol but not (R)(+)citronellol could inhibit the enzymaticactivity. FIG. 3 shows that the difference between enantiomers ofcitronellol on AChE activity is significant. The different enantiomersof citronellol were incubated with human erythrocytes' acetylcholinesterase (25 mU/mL). The enzymatic activity was determined using acommercially available kit (acetyl cholinesterase fluorescent activityassay kit, Arbor assays).

Example 3 P. graveolens Constituents that does not Inhibit AcetylCholinesterase Activity

In FIG. 4, different constituents of geranium oil (Geraniol, Citronellol(+) and Citronellol formate) were incubated with human erythrocytes'acetyl cholinesterase (25 mU/mL). The activity of acetyl cholinesterasewas determined using a commercially available kit (acetyl cholinesterasefluorescent activity assay kit, Arbor assays). According to FIG. 4, noneof the constituents geraniol, Citronellol (+) and citronellol formatecould inhibit AChE activity.

Example 4 Effect of Geranium Oil on Body Weights of MPTP-PD Mice

Body weight of MPTP-PD mice was statistically significant lower thanthat of control healthy mice. Treatment with geranium oil blocked thereduction in body weight observed in MPTP-PD mice. FIG. 5 illustrateseffect of geranium oil on the body weights of MPTP PD mice. Body weightswere recorded prior to dosing and every day during dosing, throughoutthe entire study.

MPTP formulation: Dissolve 100 mg MPTP in 5 ml of water for injectionand use this stoke solution for MPTP (20 mg/ml) dose level preparation;Dose volume: 10 ml/kg and Dose level: 30 mg/kg; Dilute stock MPTPsolution (20 mg/ml) to 4 mg/ml solution with water; Prepare sufficientvolume for each group; Prepare sufficient volume for each group.

Test Items and Vehicle formulation: Stock solution of the test item 1000mg/ml; Dilute the test item stock solution as follows: Prepare 4 ml of20 mg/ml by adding 80 μl stock solution into 3.92 ml MCT and mix welland prepare 2 ml of 50 mg/ml adding 0.1 ml stock solution into 1.9 mlMCT and mix well.

Example 5 Effect of Geranium Oil on Rota Rod (RR) Performance of MPTP PDMice

The Rota Rod (RR) performance used, reflects motor coordination, ofmice. All animals started at 3 rpm at 0 min and accelerated from 4 to 20rpm over 5 min with each mouse experiencing three trials per day with anintertrial interval of 10 min. The average latency of each mouse wascalculated. All advancements were according to the set time lines andwere approximated. The latency time to fall was measured.

A statistically significant reduction in the time spent on the RR on day11 of the study, was observed in the MPTP PD mice that were treated withvehicle, compared to control healthy mice (FIG. 6A). This observationwas also supported by a similar statistically significant reduction inthe distance traveled on the RR. (FIG. 6B). Treatment with geranium oilsignificantly increased RR latency and distance of MPTP PD mice,compared to vehicle treated MPTP PD mice (FIGS. 6A and B).

FIG. 6 demonstrates the effect of geranium oil on Rota-Rod latency anddistance of MPTP PD mice. Mice were trained on Rota-Rod, 3 trials/day,at days (−7), (−5) and (−3), and were tested at day 11 for their RRperformance. Results were analyzed using two-way ANOVA repeatedmeasurements, post hoc Bonferroni post tests. *** (p<0.001), **(p<0.01).

Example 6 Effect of Geranium Oil on Social Recognition Test in MPTP PDMice

Both MPTP-induced PD mice that were treated with geranium oil andcontrol healthy mice showed a social memory effect by spending less timeinvestigating the same juvenile animal following subsequent exposure. Incontrast, the investigative duration of the vehicle-treated MPTP PD micewas significantly higher following the subsequent juvenile exposure,compared to the control healthy mice and to geranium oil treated MPTP PDmice. Interaction ratio, reflecting social recognition memoryimprovement, of vehicle-treated MPTP PD animals was significantlyhigher, compared to healthy control mice and to MPTP PD mice that weretreated with geranium oil (FIG. 7).

The social recognition test measures memory on the basis of olfactorycues. FIG. 7 illustrates the effect of geranium oil on socialrecognition of MPTP-induced Parkonson's mice. The mouse was individuallyhoused 1 h before starting the test to isolate their odors. An adultmouse and a single juvenile mouse were placed in an arena for a periodof 5 min. During the first (T−1) interactive trial, the adult exhibitsinvestigative behavior that includes close following, sniffing orgrooming the younger mouse. The amount of time that the adult spentinvestigating the younger mouse was scored by investigator with stopclocks in seconds. The juvenile and the adult mice were then removed andreturned to their cages. Second (T−2) interactive trial was conducted120 min later in the same arena, and investigative behavior of the adultmouse was again monitored and recorded. A single memory test trial wasperformed on day 10. Recognition ratios of time spent investigating thefamiliar juvenile in T−2 divided by the time spent investigating thejuvenile in T−1 was calculated (ratio D11/2 h). Results were analyzed bytwo-way ANOVA repeated measurements, post hoc Bonferroni post tests. ***p<0.001.

Example 7 Effect of Treatment with Geranium Oil on the Number ofDopaminergic Neurons in the Substantia Nigra

FIG. 8 shows the effect of geranium oil on the number of dopaminergicneurons in the substantia nigra. Brains of all mice from (i) controlhealthy mice (ii) MPTP PD mice and (iii) MPTP PD mice that were treatedorally with 500 mg/kg geranium oil, were fixed, sectioned, immunostainedand the number of dopaminergic neurons in the substantia nigra wascounted. As can be seen in FIG. 8, the number of dopaminergic neurons inthe sunstantia nigra was markedly reduced in brains of the MPTP PD micein comparison to control healthy mice. However, treatment of MPTP PDmice with geranium oil prevented the observed neuronal cell death. Uponstudy termination on day 12, mice were euthanatized with CO₂asphyxiation. All brains were rapidly removed and stored in 10% bufferedformalin solution. Three fields were sampled from each mouse. One wayanalysis of variance was employed to compare between the differentgroups. Vehicle means the solvent with which the geranium oil wasdiluted; in this case—MCT.

All patents, patent publications, and non-patent publications cited areincorporated by reference herein.

What is claimed is:
 1. The use of effective amount of any one of thePelargonium graveolens constituents selected from a group consisting of:(S)(−)citronellol, linalool, menthone and isomenthone or any combinationthereof, in the preparation of a medicament for treating a mammalsuffering from or susceptible to a neurodegenerative condition which canbe improved or prevented by inhibition of acetylcholinesterase (AChE).2. The use of claim 1, wherein (S)(−)citronellol is the sole activeingredient.
 3. The use of claim 1, wherein linalool is the sole activeingredient.
 4. The use of claim 1, wherein menthone is the sole activeingredient.
 5. The use of claim 1, wherein isomenthone is the soleactive ingredient.
 6. The use according to claim 1, wherein thePelargonium graveolens constituents are in the form of essential oil. 7.The use according to claim 1, wherein said neurodegenerative conditionis selected from the group consisting of Alzheimer's disease,Parkinson's disease, parkinsonian dementia (PDem), Parkinson's diseasedementia (PDD), dementia with Lewy bodies (DLB), anorexia nervosa,traumatic brain injury (TBI), and Parkinson's disease falling short ofdementia (CIND-PD).
 8. An effective amount of any one of Pelargoniumgraveolens constituents selected from a group consisting of:(S)(−)citronellol, linalool, menthone and isomenthone or any combinationthereof, for use in treating a mammal suffering from or susceptible to aneurodegenerative condition which can be improved or prevented byinhibition of acetylcholinesterase (AChE).
 9. The Pelargonium graveolensconstituents of claim 8, wherein (S)(−) citronellol is the sole activeingredient.
 10. The Pelargonium graveolens constituents of claim 8,wherein linalool is the sole active ingredient.
 11. The Pelargoniumgraveolens constituents of claim 8, wherein menthone is the sole activeingredient.
 12. The Pelargonium graveolens constituents of claim 8,wherein isomenthone is the sole active ingredient.
 13. The Pelargoniumgraveolens constituents according to claim 8, wherein saidneurodegenerative condition is selected from the group consisting ofParkinson's disease, parkinsonian dementia (PDem), Parkinson's diseasedementia (PDD), dementia with Lewy bodies (DLB), anorexia nervosa,traumatic brain injury (TBI), and Parkinson's disease falling short ofdementia (CIND-PD).
 14. A pharmaceutical composition comprising thePelargonium graveolens constituents according to claim
 8. 15. Apharmaceutical composition comprising the Pelargonium graveolensconstituents according to claim 8, wherein said composition in the formof drug, food, medicinal food, food additive or beverage. 16.Pelargonium graveolens for use in treating a neurodegenerative conditionselected from the group consisting of Parkinson's disease, parkinsoniandementia (PDem), Parkinson's disease dementia (PDD), dementia with Lewybodies (DLB), anorexia nervosa, traumatic brain injury (TBI), andParkinson's disease falling short of dementia (CIND-PD).
 17. Pelargoniumgraveolens according to claim 16 for use in treating Parkinson'sdisease.
 18. Pelargonium graveolens according to claim 16, wherein saidPelargonium graveolens is the sole active ingredient.
 19. Use ofPelargonium graveolens in the preparation of a medicament for treating aneurodegenerative condition selected from the group consisting ofParkinson's disease, parkinsonian dementia (PDem), Parkinson's diseasedementia (PDD), dementia with Lewy bodies (DLB), anorexia nervosa,traumatic brain injury (TBI), and Parkinson's disease falling short ofdementia (CIND-PD).
 20. A pharmaceutical composition comprisingPelargonium graveolens for use in treating a neurodegenerative conditionselected from the group consisting of Parkinson's disease, parkinsoniandementia (PDem), Parkinson's disease dementia (PDD), dementia with Lewybodies (DLB), anorexia nervosa, traumatic brain injury (TBI), andParkinson's disease falling short of dementia (CIND-PD).
 21. Apharmaceutical composition comprising Pelargonium graveolens for use intreating Parkinson's disease.
 22. A pharmaceutical compositioncomprising Pelargonium graveolens for use in preventing death ofdopaminergic neurons.
 23. The use according to claim 5, wherein thePelargonium graveolens constituents are in the form of essential oil.24. The use according claim 5, wherein said neurodegenerative conditionis selected from the group consisting of Alzheimer's disease,Parkinson's disease, parkinsonian dementia (PDem), Parkinson's diseasedementia (PDD), dementia with Lewy bodies (DLB), anorexia nervosa,traumatic brain injury (TBI), and Parkinson's disease falling short ofdementia (CIND-PD).
 25. The Pelargonium graveolens constituentsaccording to claim 12, wherein said neurodegenerative condition isselected from the group consisting of Parkinson's disease, parkinsoniandementia (PDem), Parkinson's disease dementia (PDD), dementia with Lewybodies (DLB), anorexia nervosa, traumatic brain injury (TBI), andParkinson's disease falling short of dementia (CIND-PD).
 26. Pelargoniumgraveolens according to claim 17, wherein said Pelargonium graveolens isthe sole active ingredient.